Role of klotho and fibroblast growth factor 23 in arterial calcification, thickness, and stiffness: a meta-analysis of observational studies

This meta-analysis was conducted to clarify the role of klotho and fibroblast growth factor 23 (FGF-23) in human arterial remodeling across recent studies, in terms of arterial calcification, thickness, and stiffness. A systematic literature search was conducted on five databases for articles up to December 2023. Arterial calcification, thickness, and stiffness were determined using the calcification score and artery affected, carotid intima–media thickness (CIMT), and pulse wave velocity (PWV), respectively. Sixty-two studies with a total of 27,459 individuals were included in this meta-analysis. Most studies involved chronic kidney disease patients. Study designs were mostly cross-sectional with only one case–control and nine cohorts. FGF-23 was positively correlated with arterial calcification (r = 0.446 [0.254–0.611], p < 0.0001 and aOR = 1.36 [1.09–1.69], p = 0.006), CIMT (r = 0.188 [0.02–0.354], p = 0.03), and PWV (r = 0.235 [0.159–0.310], p < 0.00001). By contrast, Klotho was inversely correlated with arterial calcification (r = − 0.388 [− 0.578 to − 0.159], p = 0.001) and CIMT (r = − 0.38 [− 0.53 to − 0.207], p < 0.00001). In conclusion, FGF-23 and Klotho were associated with arterial calcification, thickness, and stiffness, clarifying their role in arterial remodeling processes.


Statistical analysis
Each Spearman or Pearson correlation coefficient (r) was converted to a Z-value via Fisher's transformation, which was approximately normally distributed 35,36 .The standard error of Z was calculated, and Z-values were converted via inverse Fisher's transformation to generate r and 95% CI.The extracted ORs with 95% CIs were pooled to generate the overall adjusted ORs.Pooled standardized mean difference (SMD) and 95% CI were generated to analyze the difference in the Klotho or FGF-23 level between groups with and without arterial calcification.
The chi-squared test and I 2 statistics was used to determine heterogeneity across studies.All analyses were pooled using a random-effects model.Sensitivity analysis was performed to guarantee the consistency of the results by omitting several factors that could influence the results (e.g., children and population aside from chronic kidney disease [CKD]).A one-leave-out sensitivity analysis was also performed by removing individual studies.If substantial heterogeneity occurred, subgroup analysis was employed to find the sources of heterogeneity.Publication bias was assessed visually through funnel plot asymmetry.In all analyses, a p-value of < 0.05 was considered statistically significant.Review Manager 5.4 (Cochrane Collaboration, London, UK) was used for this meta-analysis.

Study characteristics
The PRISMA flow diagram of the study selection process is shown in Fig. 1.In total, 51,534 eligible studies were documented from the searched electronic databases.Of the total articles, 31,039 were removed using automation filter tools from each database.Then, 2369 were removed for being duplicates, leaving 18,126 articles for further evaluation.Subsequently, 17,872 articles were excluded based on their titles and abstracts, whereas 254 papers were sought for retrieval.Another 22 articles were rejected for being conference abstracts and posters or having www.nature.com/scientificreports/unavailable full-texts, leaving 232 articles for full-text article review.After full-text evaluation, 176 studies were further excluded because of irrelevant outcomes, incomplete data, non-English language, irrelevant study design, and similar study/sample.In addition, 22 extra records were identified from the website and reference list search.
After judging the eligibility of the reports, 16 articles were excluded due to irrelevant outcomes, incomplete data, and similar study/sample.Ultimately, 62 articles were included in this meta-analysis.Sixty-two publications, involving 27,459 participants, were eligible according to the inclusion and exclusion criteria.The primary features of the included studies are shown in Table 1.All included studies had an observational study design.In terms of continental regions, the majority of these 62 studies are from Asia (n = 29), including China (n = 12), followed by Europe (n = 16), America (n = 8), Africa (n = 8), and Australia (n = 1).Most studies have adult participants (aged ≥ 18 years), except for three studies involving children and adolescents.The majority of the participants had CKD (n = 46).Most of the studies had cross-sectional designs (n = 50), whereas the rest were cohort and case-control studies (n = 11 and n = 1, respectively).The measured arteries varied, with mostly focused on coronary, aorta, and carotid arteries.The arterial calcification score was measured either with computed tomography (CT) or X-ray imaging, except for the studies by Milovanova 37 and Di Lullo 38 which used echocardiography.On the contrary, CIMT, and PWV were mostly measured by ultrasonography.According to the sample for FGF-23/Klotho measurement, all studies used blood sample, either in the form of plasma or serum.Forty-eight studies used serum sample, while the rest used plasma.Most FGF-23/Klotho used enzyme-linked immunoassay (ELISA) method, except for one study which used Luminex and one study did not mention the method used.Four studies did not mention the ELISA kit used.Among ELISA kit used for FGF-23 analysis, Immunotopics were used the most (36%), followed by Kainos (30%), Elabscience (8%), and Millipore (6%).As for Klotho analysis ELISA kit, Immuno-Biological Laboratories were mostly used (50%), followed by Cusabio (27.78%).

Quality assessment
The quality of the 62 included studies was assessed using the NOS, which was suitable for each study design.Among those studies, only one study 46 was considered to have low quality, 33 as moderate quality, and 28 as high quality.The quality assessment of each study using the NOS critical appraisal checklist is listed in Tables S3-S5.

Correlations between FGF-23 levels and arterial calcification
In sixteen studies, a moderate correlation was found between the FGF-23 level and arterial calcification [pooled r = 0.446 (0.254-0.611), p < 0.0001] (Fig. 2A).After sensitivity analysis by including CKD-only population (all in severe stage), cross-sectional study design, diagnosis of arterial calcification by CT, and high-quality studies, the results did not change much.However, when we perform sensitivity analysis for suspected coronary artery disease (CAD) only and diagnosis of arterial calcification by X-rays, the pooled correlations were given by r = 0.207 (CI = 0.1-0.31,n = 2, p-value 0.0002) and r = 0.282 (CI = 0.02-0.508,n = 5, p-value = 0.03), respectively.The correlation remains statistically significant at the 5% significance level, but the pooled r is lower than the correlation in the previous pooled analysis.In addition, we did not conduct sensitivity analysis for adults only since all studies regarding correlations between FGF-23 levels and arterial calcification score took adults patients only.

Correlation between the FGF-23 level and the CIMT or PWV
Eight studies reported a weak correlation between the FGF-23 level and CIMT.In the pooled analysis, the FGF-23 level positively correlated with CIMT [pooled r = 0.188 (0.02-0.354), p = 0.03] (Fig. 2B).Analysis of the correlation between the FGF-23 level and PWV also showed a significant positive correlation [pooled r = 0.235 (0.159-0.310), p < 0.00001] (Fig. 2C), in which all included studies involved CKD patients.The sensitivity analysis excluded children and included studies with severe CKD-only; however, the results were still consistent.

Correlation between the Klotho level and arterial calcification or CIMT
In contrast to FGF-23, an inverse correlation was found between the Klotho level and arterial calcification [pooled r = − 0.388 (− 0.578 to − 0.159), p = 0.001] (Fig. 2D).However, after including high-quality studies in the analysis, the pooled r changed [− 0.159 (− 0.264 to − 0.05), p = 0.005] along with reduced heterogeneity (47%).A significant negative correlation was also found between the Klotho level and CIMT [pooled r = − 0.38 (− 0.53 to − 0.207), p < 0.00001] (Fig. 2E).After including studies with the CKD-only population and high-quality studies only, the results remained stable.A meta-analysis for the correlation between the Klotho level and PWV was not performed as there was not enough number of studies that reported the correlation.

Association between the FGF-23 and arterial calcification
Seven studies have reported ORs/beta and CIs for the association between the FGF-23 level and arterial calcification generated using multivariate linear regression, and nine reported using a logistic regression model.The  3A). www.nature.com/scientificreports/For the logistic regression for the association between the FGF-23 level and arterial calcification, the pooled aOR was 1.22 (1.07-1.39)(p = 0.003) (Fig. 3B).In the sensitivity analysis that included CKD-only population and high-quality studies only, the results remained stable for both linear and logistic regression models.We did not perform pooled aOR analysis for Klotho due to limited data and varied concept of analysis between studies.

FGF-23 level in groups with arterial calcification and arterial thickness
An analysis of pooled SMD was also performed by comparing FGF-23 and Klotho levels between groups with and without arterial calcification.The group with arterial calcification had significantly higher FGF-23 levels than the group without arterial calcification [pooled SMD = 0.6 (0.36-0.84), p < 0.00001] (Fig. 4A).After conducting sensitivity analysis by including CKD-only population, measurement of calcification by the Agatston score or Kauppila index only, coronary artery only, and high-quality studies only, the results remained consistent.In subgroup analysis, the results of studies involving mild to moderate CKD only and severe CKD only also yielded consistent results.By comparing FGF-23 level difference between the groups with and without arterial thickness, the FGF-23 level was also significantly higher in the group with arterial thickness [pooled SMD = 1.26 (0.36-2.17), p = 0.006] (Fig. 4B).

Klotho level in groups with arterial calcification and arterial thickness
Two studies 68,84 have reported Klotho level differences between the groups with and without arterial calcification.However, a significant difference in Klotho levels was not found between the two groups [pooled SMD = − 0.04 (− 0.33 to 0.24), p = 0.76] (Fig. 4C).Meanwhile, a significantly lower Klotho level was found in the group with arterial thickness [pooled SMD = − 1.63 (− 3.11 to − 0.15), p = 0.03] (Fig. 4D).Sensitivity analysis revealed that the study by Castelblanco et al. 88 had a significant effect on heterogeneity.After removing this study, the pooled SMD was − 2.27 (− 2.82 to − 1.72) (p < 0.00001), and the I 2 was 49%.All analyses are summarized along with their sensitivity analyses in Table 2 for FGF-23 and Table 3 for Klotho.

Publication bias
Publication bias analysis using Funnel plot (Supplementary materials) indicates no publication bias for most analyses, except for pooled aOR of association between FGF-23 and arterial calcification in the linear regression model.However, after the study by Lee et al. 48was removed as an outlier, the funnel plot yielded a more symmetrical distribution without changing the pooled analysis.For analyses with a small number of included studies, publication bias analysis was not performed since the funnel plot and Egger's test are not recommended for less than 10 studies 91 .

Discussion
To the best of our knowledge, this study is the first meta-analysis that establishes the association of protein FGF-23 and Klotho with arterial calcification, thickness, and stiffness, and includes thorough sensitivity analyses.Our study indicates a significant positive correlation between FGF-23 and arterial calcification, CIMT, and PWV, and significant negative correlation between Klotho and arterial calcification and CIMT.FGF-23 and Klotho were also associated with arterial calcification.FGF-23 level was significantly higher in the groups with arterial calcification or thickness than in the group without arterial calcification or thickening.Furthermore, a significantly lower Klotho level was found in the arterial thickness group, not in the arterial calcification group, because only two studies were analyzed in the latter group.As stated before, arterial thickness, calcification, and stiffness is a sequential process of arterial remodeling [1][2][3][4][5] .This sequential process is affected by the FGF-23/Klotho axis 14,15 .Although Klotho itself mainly acts as the cofactor of FGF-23, its expression is downregulated by FGF-23 19,92 .In the case of vascular Klotho deficiency, FGF-23  www.nature.com/scientificreports/may induce the phenotype switching of contractile VSMCs to synthetic VSMCs mediated by FGF receptor-1 (FGFR-1) and Erk1/2 phosphorylation along with an increase in proliferation, which further induces thickening, and stiffening of the arterial wall 93 .This was confirmed in our study, which showed higher FGF-23, and lower Klotho levels in the arterial remodeling process.FGF-23 and Klotho also have contradictory effects on NO production.Klotho may revert the FGF-23-induced vasoconstriction by increasing NO production to dilate the arteries 93,94 .Furthermore, atherosclerotic plaques that reside in the arterial wall show a stronger FGFR signaling in response to FGF-23 and a lower expression of contractile VSMC phenotype 95 .The stronger FGFR signaling can cause further Klotho deficiency caused by FGF-23-induced Klotho downregulation.Interestingly, FGF-23, and Klotho have a unique or special affinity to FGFR-1 94,96 .The binding of Klotho to the principal effector site of FGFR-1 may induce the phosphaturic effects of FGF-23 on the kidney.Thus, the Klotho/FGFR-1/FGF-23 complex in the kidney is an important signaling pathway, either in generating, or counteracting hyperphosphatemia 94 .Hyperphosphatemia is avoided in this process because of its strong effect on inducing vascular calcification 97 .Therefore, all of these processes induce arterial remodeling, including vascular calcification, thickening, and stiffening.
Interestingly, the positive effect sizes of FGF-23 in vascular calcification and CIMT were stronger in the CKDonly subgroup analyses than in the overall analyses.Additionally, the pooled correlation between FGF-23 level and CIMT was also stronger in severe CKD only group than in all CKD group, albeit the number of studies was lower.This was further supported by a stronger negative correlation of Klotho to vascular calcification of the CKD-only study population; however, this was not seen in CIMT because only two studies analyzed Klotho in CKD.Despite these findings, we acknowledged that most of our included studies involved CKD patients.One could argue that there might be a tendency toward a significant finding, where higher FGF-23 and lower Klotho levels were associated with the conditions, due to the populations being predominantly CKD.Nevertheless, we observed that this is not utterly the case.For example, in the forest plot of the pooled correlation between FGF-23 and arterial calcification (Fig. 2A), studies with CKD and non-CKD-only populations presented with varying directions of effect sizes.Studies by Cianciolo 45 and Nitta 30 that included only CKD patients showed a negative direction of effect sizes.Meanwhile, studies by Masai 49 and Morita 31 showed a positive direction of effect sizes despite including non-CKD populations (suspected CAD patients).This finding was confirmed by our sensitivity analysis including only these two studies which still showed a significant positive effect size, although it was lower than that of the analysis with only CKD patients.In Klotho analyses, we could observe such similar cases, in which studies with non-CKD populations showed a negative direction of effect sizes, i.e., Koga 61 and Morita 31 in Fig. 2D and Jeong 63 , Keles 64 , and Keles 65 in Fig. 2E.These findings indicated that FGF-23 and Klotho play important roles as a promoter and inhibitor, respectively 98 , in both CKD and non-CKD patients, and are not being entirely affected by kidney function status.
We also found a stronger FGF-23-CIMT correlation when two studies including children with CKD were excluded from the analysis.Two reasons could explain this interesting finding.First, despite having CKD, the pediatric populations were still in the growth and development phase, including their vascular thickness.The development of vascular thickness is ongoing throughout life; therefore, the vascular thickness might not be early seen 99 .Second, the number of children with CKD in the two studies was very limited compared with the number of adult patients in another five studies.Furthermore, the FGF-23-PWV correlation did not change much in the subgroup analyses excluding children and CKD-only participants.An interesting fact was stated by London 100 ; i.e., the result of PWV measurement was age-and blood pressure-dependent.This might not change the correlation strength of FGF-23 and PWV because children and patients with CKD had an individual range of blood pressure.
Despite our findings, this study has four main limitations.First, the definitions, and parameters used for assessing arterial calcification, thickness, and stiffness vary.For example, several studies inappropriately analyzed arterial calcification using CIMT or PWV.CIMT was only designed for measuring the extent of the intimal and medial layers of the carotid arterial wall 101 , whereas PWV was only designed for measuring velocity and distensibility through the transmitted pulse wave in the arterial system 102 .Based on the latter statement, both CIMT and PWV did not measure the degree of calcification in the arterial wall, only the extent, and distensibility of the arterial wall, respectively.However, we overcame this limitation by classifying the analyses of calcification, thickness, and stiffness based on the assessment method used in each study: (1) calcification score to determine arterial calcification, (2) CIMT to determine arterial thickness, and (3) PWV to determine arterial stiffness.Second, the heterogeneities among the included studies were appreciable because of several factors, including study design, type of the analyzed artery, assessment process, sample size, age, and population type.We have also performed subgroup analyses to minimize the bias that might be caused by this limitation.We also have tried to explore the cause of the heterogeneity, i.e., measurement method used.However, all sample used blood specimen and almost all study used ELISA method.Hence, the heterogeneity might not likely be caused by the measurement method.Third, there was no detailed data regarding FGF-23 and Klotho levels in each CKD stage.There were limited studies which recruited participants from mild to moderate CKD only, since most included studies used HD or advanced stage CKD as their participants.Nevertheless, we have tried to do subgroup analysis for the available data to minimize this limitation, in which we proved that FGF-23 levels were significantly increased in arterial calcification, either in mild-to-moderate or severe CKD group.Lastly, considering that all included studies had an observational design investigating only associations, the true causality between FGF-23/Klotho and arterial calcification, thickness, and stiffness still cannot be discerned.Moreover, despite of the limitations, this meta-analysis could provide a useful insight on the role of FGF-23 and Klotho in arterial remodeling, since the underlying remodeling process is relatively complex and a unified conclusion is needed.Further research is warranted to establish the role of FGF23 and Klotho in clinical practice.We also suggest preclinical studies to explore further about the exact mechanism of FGF23 and Klotho on arterial remodeling process.

Figure 1 .
Figure 1.PRISMA flow diagram of the literature search.

Figure 2 .
Figure 2. Forest plot of the pooled r for the correlation between: (A) FGF-23 level and arterial calcification; (B) FGF-23 level and CIMT; (C) FGF-23 level and PWV; (D) Klotho level and arterial calcification; (E) Klotho level and CIMT.All analyses are pooled using a random-effects model.

Figure 3 .
Figure 3. Forest plot of the pooled OR for the association between: (A) FGF-23 level and arterial calcification in linear regression model and (B) FGF-23 level and arterial calcification in logistic regression model.All analyses are pooled using a random-effects model.

Figure 4 .
Figure 4. Forest plot of the pooled SMD for: (A) FGF-23 level in calcification/no calcification groups; (B) FGF-23 level in high CIMT/low CIMT groups; (C) Klotho level in calcification/no calcification groups; (D) Klotho level in high CIMT/low CIMT group.All analyses are pooled using a random-effects model.

Table 2 .
Summary of meta-analysis of FGF-23 with each sensitivity analysis.CKD chronic kidney disease, RE random effect, SMD standardized mean difference.

Table 3 .
Summary of meta-analysis of Klotho with each sensitivity analysis.CKD chronic kidney disease, RE random effect, SMD standardized mean difference.